JPH0462022B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method and apparatus for dynamically measuring glucose concentration by measuring current value using an enzyme electrode that detects an enzyme reaction.

Prior Art Various methods are known for measuring the concentration of glucose and the like using enzymatic reactions. For example, there is a method of reacting with oxygen using glucose oxidase and measuring the amount of decrease in dissolved oxygen at that time using an oxygen electrode (published by Kodansha, introduced in ``Basics and Applications of Chemical Sensors'', edited by Tetsuro Shimizu and others), Alternatively, there is a method of measuring the generated hydrogen peroxide using a hydrogen peroxide electrode. That is, glucose is oxidized by glucose oxidase and decomposed into gluconolactone and hydrogen peroxide. The amount of glucose can be measured by measuring the oxygen consumed or the hydrogen peroxide produced by this reaction using an oxygen electrode or a hydrogen peroxide electrode.

The principle is shown in Figure 2.

In Figure 2, 35 is an enzyme membrane, 36 is a Teflon membrane, 3
7 indicates a cathode.

A portion of the oxygen that diffuses from the glucose solution to the electrode is consumed by the immobilized glucose oxidase membrane, reducing the oxygen concentration reaching the electrode.

As a result, the current value at the electrode also decreases. However, over time, an equilibrium is established between the amount of oxygen consumed by the enzyme membrane and the amount of oxygen diffused from the solution, and a steady state is reached at a certain current value. This current value depends on the glucose concentration in the solution.

Therefore, a glucose solution of unknown concentration can be measured by measuring the current value of each glucose solution whose concentration value has been measured in advance and creating a calibration curve.

The above is a method of measuring dissolved oxygen using an electrode, but there is also a method of colorimetrically quantifying hydrogen peroxide produced by the action of glucose oxidase on glucose using peroxidase and a coloring reagent such as O-toluidine. be.

In addition, a device was developed in 1987 that allowed blood to flow together with a buffer solution through a measuring section having an enzyme electrode as described above, and then brought the blood into contact with the electrode in a flowing state to measure the sugar content.
It is known from Publication No. 54847.

Problems to be Solved by the Invention However, the former method using enzymes can only be used in batches and off-line. There were problems with automation in practical use, such as the need to dilute the sample appropriately and the need for manual sample feeding. The latter publication is online, but when measuring a sample with a constant glucose concentration, only constant values are always obtained for the amount of oxygen and current generated by the enzyme reaction. Therefore, it is not possible to accurately measure samples over a wide concentration range.

Means for Solving the Problems Therefore, the technical problem of the present invention is to provide a method and device for measuring glucose, which can automatically and directly measure glucose concentration on-line and can accurately measure samples over a wide concentration range. The technical means of the present invention to solve this technical problem is to reduce the amount of oxygen consumed or the amount of hydrogen peroxide produced by the enzyme reaction using an oxygen electrode or The hydrogen electrode converts the electricity into electricity, and the glucose concentration is measured using the electricity. A fixed amount of sample is intermittently poured into a reaction cell equipped with an enzyme electrode, with a buffer solution constantly flowing. The glucose concentration is measured from the amount of current change due to the enzyme reaction when the sample passes through the reaction cell.Glucose is measured by changing the contact time between the sample and the enzyme by adjusting the flow rate of the buffer solution. An apparatus for carrying out the above method is one that connects a reaction cell equipped with an enzyme electrode with a buffer tank that is constantly heated to a constant temperature through a conduit, and is equipped with an automatic sample quantitative feeding device. It is connected to the conduit via a switching valve.

Effects of the Invention According to this technical means, glucose concentration can be measured by intermittently feeding a fixed amount of sample into a state where a buffer solution is constantly flowing, and the glucose concentration can be measured by By changing the contact time of the sample by adjusting the flow rate of the buffer solution, the measurement range of the sample can be adjusted. Therefore, even with highly concentrated glucose solutions, the dilution work can be eliminated, and continuous measurement can be performed automatically in an online method. There is a characteristic that
In other words, the amount of oxygen consumed can be controlled by controlling the contact time between the enzyme and the sample, and the amount of current can also be controlled, so when measuring a sample with a high glucose concentration, the flow rate can be increased to shorten the contact time with the enzyme. By suppressing the amount of oxygen consumed, measurements can always be made in the linear range of the calibration curve.For low-concentration samples, on the other hand, the flow rate can be controlled to prolong the reaction time with the enzyme, allowing measurements to be made in the linear range of the calibration curve. By controlling such enzymatic reactions, samples with a wide concentration range can be measured with high precision. When diluting by varying the injection volume, a highly concentrated sample with a large dilution rate may cause an error, but in the present invention, the flow rate of the buffer solution is controlled, a calibration curve is set according to the flow rate, and the flow rate is adjusted. Since the calibration curve is switched according to the current value and the glucose concentration is measured from the current value, there is no dilution error and there is an advantage that accurate measurement can be performed over a wide range.

Example The embodiments shown in the drawings will be described below.

In FIG. 1, 1 is a column filled with, for example, an immobilized enzyme for decomposing lactose (immobilized lactase), and decomposed milk is discharged as a product from an outlet 2.

3 is the branch pipe from the pump 4 to the T-shaped joint 3
2. Communicates with an autopipette 6 via a switching valve 5. Excess product is discharged via conduit 33.

The standard solution tank 7 also communicates with the autopipette 6 via a switching valve 5 through a conduit 8.

Air is sent into the buffer solution in the buffer solution tank 14 through a conduit 10 to bring the amount of dissolved oxygen into a saturated state, and the buffer solution is heated by a heater 11, and its temperature is controlled by a controller 13 via a sensor 12. .

The buffer solution in the tank 14 is supplied by a peristaltic or metering pump 15 via a conduit 16 to a glucose sensor 18 in a constant temperature bath 17.

Further, the sample or standard solution aspirated into the autopipette 6 is passed through the supply valve 19 to the conduit 16.
It is connected to the.

By switching the supply valve 19, therefore, a fixed amount of sample or standard solution can be supplied to the buffer solution constantly flowing in the conduit 16. Further, the sample or standard solution other than when being measured is discharged from the conduit 34.

The glucose sensor 18 is as shown in FIG. 3, with reference numeral 38 an oxygen electrode, 39 a Teflon membrane, 40 an enzyme membrane, 41 a cellulose dialysis membrane, 42 a cell, and 43 an O-ring. The buffer solution constantly flows through the passages 44, 45, and 46 of the reaction cell.

In FIG. 1, the current value detected by the glucose sensor 18 is amplified by the amplifier 26, and the device 27
Noise is removed, and the glucose concentration is calculated by the computer 28 and measured. In addition, 29
indicates the output printer.

Now, switch the sample and standard solution at the appropriate time using the switching valve 5, aspirate a certain amount using the autopipette 6, and then
Send to 9.

Buffer is pumped by metering pump 15 through T-shaped fitting 31 into the reaction cell of glucose sensor 18 and discharged through tube 30. The supply valve 19 is switched while the buffer solution is constantly flowing, and a fixed amount of the sample or standard solution is injected using the autopipette 6.

The method of measuring the concentration from the indicated value of the electrode is to aspirate a glucose solution of a known concentration from the conduit 8 with the autopipette 6 and inject it through the T-shaped fitting 31.

Then, the relationship between the current change when passing through the reaction cell of the sensor 18 and the glucose concentration is determined, and using this relationship, the computer 2 calculates the indicated value obtained when the sample is similarly injected from the conduit 3.
Measure the glucose concentration at step 8.

Here, when determining the relationship between the amount of change in current when passing through the reaction cell of the sensor 18 and the glucose concentration, the following results were obtained.

In other words, as mentioned earlier, the current value depends on the glucose concentration due to the amount of oxygen consumed that diffuses from the glucose solution to the electrode through the enzyme membrane, or from the decreasing oxygen concentration and the resulting decrease in the current value at the electrode. However, in the experiment, we used a glucose sensor as an enzyme electrode, which was equipped with an immobilized enzyme membrane in which glucose oxidase was immobilized on a DEAE cellulose membrane using an ion bonding method, as described later, and attached to the sensing surface of a dissolved oxygen electrode. The buffer solution was installed in a buffer flow path that was constantly flowing, and an experiment was conducted by flowing a glucose solution with a pre-measured concentration into this flow path, and it was found that the current value varied depending on the flow rate of the buffer solution. I found out that things change.

In other words, when we measured the amount of current change in a glucose solution with a pre-measured concentration while keeping the flow rate constant, and measured the amount of current change in the same sample while changing the flow rate in the same way, we found that there was a difference between the concentration and the amount of current change. The results were shown in Figure 4.

The vertical axis in FIG. 4 is the amount of change in current value, and the horizontal axis is the concentration. The flow speeds V1, V2, V3, V4, and V5 in the table are respectively
0.38cm/sec, 1.47cm/sec, 2.92cm/sec, 6.95
cm/sec, 8.88cm/sec.

By the way, assuming that the current value when only the buffer solution was passed was 100%, the calibration curves for each flow rate within the range of 50 to 80% showed a straight line, and the slopes were the same.

Therefore, in the measurement, as shown in Figure 5, for two or more types of glucose solutions whose concentration values have been measured in advance at a flow rate Va, the amount of current change is measured for each, and the calibration curve y = alogx +
Find b. Here, x is the concentration and y is the amount of current change. Next, at this flow rate Va, the glucose solution exhibiting the amount of current change Ia in the measurement range of 80% is measured while changing the flow rate, and the flow rate Vb showing the amount of current change Ib in the measurement range of 50% is determined. Because of this, the slope of the calibration curve at the flow rate Vb is the same as the slope of the calibration curve at the flow rate Va, so Y = alogx +
b-(Ia-Ib). The equation of the calibration curve when the flow rate is changed after that is y=a logx+bn(Ia
−Ib). Note that n here is the number of calibration movements, and n=2, 3, 4, . . . .

Also, the flow rate of each calibration curve is (Vb−Va)
It is only necessary to move by n, and the formula Vx=Va+(Vb−
Va) can be found by n. n here takes the same value as n above. By using these two equations, a calibration curve as shown in FIG. 5 is created, and the respective flow rates and equations of the calibration curve are stored in the computer. Then, set the flow rate earlier so that the amount of current change y when flowing the glucose solution of unknown concentration takes a current value in the range of 50% to 80% when the current value when the buffer solution is flowing is 100%. The glucose concentration can be determined from a calibration curve by selecting one of these in stages and using the calibration curve at that flow rate.

Now, based on the above measurements, it is possible to automatically control the amount of milk in column 1 to keep the lactose decomposition rate constant, and as shown by the imaginary line in Figure 1, pump 4, autopipette 6, pump 15,
The supply valve 19 can be automatically controlled.

To describe a control method using the above-mentioned device when producing digested milk by filling a column with an immobilized enzyme for lactose-digesting milk and allowing milk to flow through column 1 at a constant flow rate, it generally becomes fixed after long-term operation. lactase activity decreases, and the decomposition rate fluctuates.

There, a fixed amount of decomposed milk is sent to a glucose sensor using a peristaltic pump, and the amount of glucose is converted into a measured electrical signal, which is sent to a computer 28. The measured electrical signal is converted into a decomposition rate in the computer 28 and compared with a set decomposition rate. If there is a difference between the measured value and the set value, adjust the flow rate of the pump (not shown) that supplies milk to the column according to the difference so that the decomposition rate of breast milk becomes the set decomposition rate. It is something to control.

A concrete implementation example is as follows. To immobilize the immobilized glucose oxidase membrane used in the glucose sensor, 10 mg of glucose oxidase (manufactured by Amano Pharmaceutical) was dissolved in 10 ml of PH6.0, 0.1M acetate buffer, and a DEAE cellulose membrane with a diameter of 6 mm was placed in the solution for 3 hours. It was immersed and fixed in a suction oven.

This immobilized membrane is used as a dissolved oxygen electrode (manufactured by Ishikawa Seisakusho).
It was placed on the detection surface of the device, covered with a dialysis membrane, and secured with an O-ring.

This enzyme electrode was attached to a flow cell.

The buffer solution is 0.1M acetic acid and 0.1M sodium acetate.
It was used after adjusting the pH to 6.0.

The measuring device used a constant temperature bath and buffer solution at 40°C, and the buffer solution flow rates were 2.08 cm/sec and 6.95 cm/sec.

The injection volume by autopipette was 0.5 ml.

The standard solution is a 5% diluted medical glucose solution.
0.6g/dl, 0.7g/dl, 0.8g/dl, 0.9g/dl,
When 1.0 g/dl was prepared and lactose-decomposed milk was used as a sample, this device was compared with the conventional method, that is, the value obtained using the YSI Sugar Analyzer, and there was almost no difference.

In any case, the present invention involves injecting a fixed amount of a sample into the reaction cell of a glucose sensor while a buffer solution is constantly flowing at a constant flow rate into the reaction cell of the glucose sensor. This method calculates the concentration from the amount of change, and the measurement range can be adjusted by changing the flow rate of the buffer solution.It is possible to accurately and easily measure high concentrations of glucose, and in particular, it can be used directly and automatically without dilution. It has the effect of being measurable.

In the examples, the substance to be detected by reaction is dissolved oxygen, but hydrogen peroxide may also be used.
Furthermore, it is preferable that the enzyme be immobilized in the form of a membrane. Furthermore, a sample containing a buffer solution and glucose is introduced and discharged through a conduit connected to the reaction cell, but it is preferable that the sample should be able to escape immediately if air is mixed in, and the buffer solution used in the example was acetic acid. There is no particular limitation as long as it does not inhibit the enzymatic reaction, and an electric autopipette is preferable.
Alternatively, a highly accurate micro-metering pump may be used.

[Brief explanation of drawings]

Fig. 1 is an explanatory diagram of the device of the present invention, Fig. 2 is an explanatory diagram showing the measurement principle, Fig. 3 is an explanatory diagram of the glucose sensor, and Fig. 4 is a diagram of the relationship between current variation and concentration.
FIG. 5 is an explanatory diagram of a calibration curve. 1... Column, 5... Switching valve, 6... Autopipette, 7... Standard solution tank, 14... Buffer solution tank, 17... Constant temperature bath, 18... Glucose sensor, 28... Computer.

Claims (1)

[Claims] 1. The amount of oxygen consumed or the amount of hydrogen peroxide produced by an enzyme reaction is converted into an amount of electricity using an oxygen electrode or a hydrogen peroxide electrode, and the glucose concentration is determined by the amount of electricity. It is something to be measured,
A fixed amount of sample is intermittently fed into a reaction cell equipped with these enzyme electrodes while a buffer solution is constantly flowing, and the glucose concentration is determined from the amount of current change due to the enzyme reaction when the sample passes through the reaction cell. 1. An automatic method for measuring glucose, characterized in that glucose is measured by varying the contact time between a sample and an enzyme by adjusting the flow rate of a buffer solution. 2. In the method for automatically measuring glucose according to claim 1, when the amount of current change due to fluctuations in the glucose concentration to be measured is 100% when only the buffer solution is passed, Obtain a calibration curve for each flow rate set in advance so that it always falls within the range of 50% to 80%, and change the flow rate so that the measured current value falls within one of the calibration curves. 1. A method for automatically measuring glucose, comprising automatically measuring a glucose concentration from a current value of a corresponding calibration curve. 3. Automatic measurement of glucose by connecting a reaction cell equipped with an enzyme electrode with a buffer tank always heated to a constant temperature through a conduit, and connecting an automatic sample quantitative feeder to the conduit via a switching valve. Device.